1. Field of the Invention
The use of enzymes finds broad application in a wide variety of analytical techniques, such as diagnostic assays, histochemical staining, chromatographic examination, and the like. In diagnostic assays, a number of assay techniques have been developed which involve steric effects for modulating the observed signal in relation to the analyte. For the most part, steric repulsions are involved, where the substrate is inhibited from approaching the active site of the enzyme by the presence of large molecules bound to the enzyme in the vicinity of the active site.
In employing enzymes in the diagnostic assays, the enzymes are normally labeled with a ligand, so that upon binding of antiligand to ligand, the substrate is sterically excluded from the active site. In chosing an enzyme for use in diagnostic assays, there are many considerations which come into play. Among these considerations are storage stability, turnover rate, presence in fluids to be measured, susceptibility to inhibition, pH optimum, susceptibility to steric exclusion of substrate, availability, degree of characterization, and methods for determination of enzymatic activity. Therefore, different enzymes may be preferable for different assays.
The glycosidases have many desirable properties, in that many of them have high turnover rates, are stable for long periods of time, particularly when lyophilized, can be readily conjugated to other molecules, and have substrates other than their natural substrates which permit the determination of enzyme activity by spectrophotometric means. However, these reagents are monosaccharides and are therefore small and are only difficultly excluded from the enzyme active site by steric effects. In order to use these enzymes where steric exclusion is applicable for modulating the signal in relation to the amount of analyte in the assay medium, it is desirable to provide substrates which would be more readily sterically excluded from the active site.
2. Description of the Prior Art
Cuatrecasas, J. Biol. Chem., 246, 196 (1971) describes an affinity chromatography column for .beta.-galactosidase employing .beta.-thiogalactoside linked to an insoluble support by a dipropylenetriamine spacer arm. U.S. Pat. No. 3,817,837 describes a homogeneous enzyme immunoassay involving steric exclusion of a substrate from an enzyme. More recently, a technique involving particulate reagents is described in copending application Ser. No. 964,099 filed Nov. 24, 1978.